Dyslipoproteinaemias are disorders of the metabolism of the lipoproteins responsible for transport of lipids such as cholesterol and triglycerides in the blood and the peripheral fluids. They lead to major pathologies associated, respectively, with hypercholesterolaemia or hypertriglyceridaemia, such as, for example, coronary artery disease.
xe2x80x9cCoronary artery diseasexe2x80x9d is a collective term for a variety of symptomatic conditions including angina, myocardial infarction, and non-specific chest, arm and face pain, which result from atherosclerosis of the arteries that supply blood to the heart.
xe2x80x9cPremature atherosclerosisxe2x80x9d as used herein refers to the clinical presentation of signs and symptoms of coronary artery disease before the age of 65.
Atherosclerosis, commonly known as xe2x80x9chardening of the arteries,xe2x80x9d is a complex disease of polygenic origin, which is defined from a histological standpoint by deposits (lipid or fibrolipid plaques) of lipids and of other blood derivatives within the wall or endothelium of the large arteries (aorta, coronary arteries, carotid). These plaques, which are more or less calcified according to the degree of progression of the process, may be coupled with lesions, and are associated with the accumulation of fatty deposits in the arteries, consisting essentially of cholesterol esters.
The plaques are accompanied by a thickening of the arterial wall, with hypertrophy of the smooth muscle, appearance of foam cells and accumulation of fibrous tissue. The atheromatous plaque protrudes markedly from the wall, endowing it with a stenosing character responsible for vascular occlusions by atheroma, thrombosis or embolism which occur in those patients who are most affected. Thus, the dyslipoproteinaemias can lead to very serious cardiovascular pathologies such as infarction, sudden death, cardiac decompensation, stroke, and the like.
Because of the significant relationship between coronary artery disease and heart attacks, considerable effort has been devoted to identifying the biochemical causes of atherosclerosis. This research has shown that high levels of total cholesterol, low density lipoprotein (LDL), very low density lipoprotein (VLDL) and triglycerides are associated with increased risk of coronary artery disease, while high levels of high density lipoproteins (HDL) are associated with decreased risk of coronary artery disease. See, Gordon et al., The Amer. J. Med. 62: 707-714 (1977). However, while observation of lipoproteins, cholesterol and triglycerides can provide a basis for identifying individuals at risk of coronary artery disease, the levels of these substances are themselves symptoms of an underlying biochemical defect which remains unidentified. Thus, specific treatment of the ultimate cause rather than an intermediate condition, and prediction of risk prior to the onset of this intermediate condition is not possible through such observation.
Studies directed towards the underlying cause of coronary artery disease have identified a number of mutations in genes coding for proteins involved in lipid transport and metabolism that appear to be associated with an increased risk. Examples include a large number of mutations in the low-density lipoprotein receptor gene, Hobbs et al., Human Mutations 1: 445-466 (1992), and a single mutation in the apolipoprotein-B (Apo-B) gene which underlies familial defective Apo-B in many parts of the world. Soria et al., Proc. Nat""l Acad Sci. USA 86: 587-91 (1989). In addition, mutations in other genes which play a significant role in HDL metabolism 20 such as the cholesterol ester transferase protein (CETP) gene, Brown et al., Nature 342: 448-451 (1989) and the gene for Apo-Al, Rubin et al., Nature 353: 265-266 (1991), have also been shown to be associated with either enhanced resistance or increased susceptibility to atherosclerosis. However, these mutations are uncommon and thus far no specific mutation in any gene has been found in a significant number (i.e.,  greater than 1%) of patients with coronary artery disease or premature atherosclerosis. Accordingly, these test results while interesting do not offer the opportunity to provide evaluation or therapy to significant numbers of patients
At the present time these pathologies, and especially the hypercholesterolaemias, are treated essentially by means of compounds which act either on cholesterol biosynthesis (hydroxymethylglutarylcoenzyme A reductase inhibitors, statins), or on the uptake and removal of biliary cholesterol (sequestering agents or resins), or alternatively on lipolysis by a mode of action which remains to be elucidated at molecular level (fibrates). Consequently, all the major classes of medicinal products which have been used in this indication (sequestering agents, fibrates or statins) are directed only towards the preventive aspect of atheromatous plaque formation and not, in fact, towards the treatment of atheroma. Current treatments of atheroma following coronary accident are merely palliative, since they do not intervene in cholesterol homeostasis and are surgical procedures (coronary bypass, angioplasty).
It has now been found that a single point mutation in the human lipoprotein lipase gene which results in an Axe2x86x92G nucleotide change at codon 291 (nucleotide 1127) of the lipoprotein lipase gene, and a substitution of serine for the normal asparagine in the lipoprotein lipase gene product is seen with increased frequency in patients with coronary artery disease, and is associated with an increased susceptibility to coronary artery disease, including in particular premature atherosclerosis. This is expressed as a diminished catalytic activity of lipoprotein lipase, lower HDL-cholesterol levels and higher triglyceride levels. Thus, in accordance with one embodiment of the present invention there is provided a method for evaluating susceptibility of a human individual to premature atherosclerosis and other forms of coronary artery disease comprising the steps of:
(a) obtaining a sample of DNA from the individual; and
(b) evaluating the sample of DNA for the presence of nucleotides encoding a serine residue as amino acid 291 of the lipoprotein lipase gene product.
The presence of a serine residue is indicative of increased susceptibility in the patient.
The invention further provides a kit for performing the method of the invention. Such a kit comprises a pair of primers selected to amplify a region of a human lipoprotein lipase gene spanning amino acid 291 of human lipoprotein lipase. Appropriate additional reagents may also be included in the kit such as polymerase enzymes, nucleoside stock solutions and the like.
A further aspect of the present invention is a method of treating patients suffering from or likely to suffer from premature atherosclerosis and other forms of coronary artery disease as a result of a lipoprotein lipase deficiency using gene therapy. This, for example, may be accomplished using adenovirus-mediated or retrovirus-mediated gene therapy, and can be performed using either an in vivo or an ex vivo approach.
Thus, the present invention also constitutes a novel therapeutic approach to the treatment of pathologies associated with dyslipoproteinaemias, which may be caused by, for example, lipoprotein lipase deficiency. It proposes an advantageous solution to the drawbacks of the prior art, by demonstrating the possibility of treating pathologies associated with dyslipoproteinaemias by gene therapy, by the transfer and expression in vivo of a gene coding for a lipase involved in lipoprotein metabolism. The invention thus affords a simple means permitting specific and effective treatment of these pathologies.
Gene therapy consists in correcting a deficiency or an abnormality (mutation, aberrant expression, and the like) or in providing for the expression of a protein of therapeutic interest by introducing genetic information into the affected cell or organ. This genetic information may be introduced either ex vivo into a cell extracted from the organ, the modified cell then being reintroduced into the body, or directly in vivo into the appropriate tissue. In this second case, different techniques exist, including various techniques of transfection involving complexes of DNA and DEAE-dextran (Pagano et al., J. Virol. 1 (1967) 891), of DNA and nuclear proteins (Kaneda et al., Science 243 (1989) 375), and of DNA and lipids (Felgner et al., PNAS 84 (1987) 7413), the use of liposomes (Fraley et al., J. Biol. Chem. 255 (1980) 10431), and the like. More recently, the use of viruses as vectors for gene transfer has been seen to be a promising alternative to these physical transfection techniques. In this connection, different viruses have been tested for their capacity to infect certain cell populations. This applies especially to retroviruses (RSV, HMS, MMS, and the like), the HSV virus, adeno-associated viruses and adenoviruses.
The present invention constitutes a novel therapeutic approach to the treatment of pathologies associated with dyslipoproteinaemias, consisting in transferring and expressing in vivo genes coding for lipases involved in lipoprotein metabolism. It is especially advantageous that applicants have now shown that it is possible to construct recombinant viruses containing a DNA sequence coding for a lipase involved in lipoprotein metabolism, and to administer these recombinant viruses in vivo, and that this administration permits a stable and effective expression of a biologically active lipase in vivo, and without a cytopathological effect.
The present invention is also the outcome of the demonstration that adenoviruses constitute especially effective vectors for the transfer and expression of such genes. In particular, the present invention shows that the use of recombinant adenoviruses as vectors enables levels of expression of these genes to be obtained which are sufficiently high to produce the desired therapeutic effect.
The present invention thus affords a novel approach to the treatment and prevention of cardiovascular and neurological pathologies associated with dyslipoproteinaemias.
A subject of the invention lies in a defective recombinant virus comprising a nucleic acid sequence coding for a lipase involved in lipoprotein metabolism.
The subject of the invention is also the use of such a defective recombinant virus for the preparation of a pharmaceutical composition intended for the treatment and/or prevention of cardiovascular diseases.
The present invention also relates to the use of cells modified genetically in vivo or ex vivo with a virus as described above, or of cells producing such viruses, implanted in the body, permitting a sustained and effective in vivo release of a biologically active lipase.